Liu Yijin, Freeman Alasdair, Déclais Anne-Cécile, Gartner Anton, Lilley David M J
Cancer Research UK Nucleic Acid Structure Research Group, The University of Dundee, Dundee, United Kingdom.
Cancer Research UK Nucleic Acid Structure Research Group, The University of Dundee, Dundee, United Kingdom.
Methods Enzymol. 2018;600:543-568. doi: 10.1016/bs.mie.2017.11.021. Epub 2018 Feb 1.
Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.
DNA中的四链霍利迪连接体是基因重组的核心中间体，必须加工成规则的双链体形式。实现这一过程的一种机制称为解离，由结构选择性核酸酶介导。GEN1是真核细胞中一种重要的连接体解离酶，属于FEN1/EXO1核酸酶超家族成员。由于人源GEN1易于聚集而难以操作，因此利用生物信息学鉴定了嗜热真菌的直系同源物，并证明其具有优异的特性。本文描述了嗜热毛壳菌中该酶的表达和纯化，以及研究其生化特性的方法。该酶与低等生物的连接体解离酶非常相似，以二聚体形式结合到连接体上，进行对称的双侧切割，其中第二次切割加速以促进有效解离。本文还描述了嗜热毛壳菌GEN1的结晶以及DNA产物复合物的结构。晶格中复合物的并置表明了具有完整连接体的二聚体酶的结构是如何组织的。
